Muscle enzyme activities in a deep-sea squaloid shark, Centroscyllium fabricii, compared with its shallow-living relative, Squalus acanthias

The activities of several enzymes of energy metabolism were measured in the heart, red muscle, and white muscle of a deep and a shallow living squaloid shark, Centroscyllium fabricii and Squalus acanthias, respectively. The phylogenetic closeness of these species, combined with their active predatory nature, similar body form, and size makes them well matched for comparison. This is the first time such a comparison has been made involving a deep-sea elasmobranch. Enzyme activities were similar in the heart, but generally lower in the red muscle of C. fabricii. Paralleling the trend seen in deep-sea teleosts, the white muscle of C. fabricii had substantially lower activities of key glycolytic enzymes, pyruvate kinase and lactate dehydrogenase, relative to S. acanthias or other shallow living elasmobranchs. Unexpectedly, between the squaloid sharks examined, creatine phosphokinase activity was higher in all tissues of the deep living C. fabricii. Low white muscle glycolytic enzyme activities in the deep-sea species coupled with high creatine phosphokinase activity suggests that the capacity for short burst swimming is likely limited once creatine phosphate supplies have been exhausted.     

Decreasing urea∶trimethylamine N-oxide ratios with depth in chondrichthyes: a physiological depth limit?

In marine osmoconformers, cells use organic osmolytes to maintain osmotic balance with seawater. High levels of urea are utilized in chondrichthyans (sharks, rays, skates, and chimaeras) for this purpose. Because of urea's perturbing nature, cells also accumulate counteracting methylamines, such as trimethylamine N-oxide (TMAO), at about a 2∶1 urea∶methylamine ratio, the most thermodynamically favorable mixture for protein stabilization, in shallow species. However, previous work on deep-sea teleosts (15 species) and chondrichthyans (three species) found an increase in muscle TMAO content and a decrease in urea content in chondrichthyans with depth. We hypothesized that TMAO counteracts protein destabilization resulting from hydrostatic pressure, as is demonstrated in vitro. Chondrichthyans are almost absent below 3,000 m, and we hypothesized that a limitation in urea excretion and/or TMAO retention might play a role. To test this, we measured the content of major organic osmolytes in white muscle of 13 chondrichthyan species caught with along-contour trawls at depths of 50-3,000 m; the deepest species caught was from 2,165 m. Urea and TMAO contents changed significantly with depth, with urea∶TMAO declining from 2.96 in the shallowest (50-90 m) groups to 0.67 in the deepest (1,911-2,165 m) groups. Urea content was 291-371 mmol/kg in the shallowest group and 170-189 mmol/kg in the deepest group, declining linearly with depth and showing no plateau. TMAO content was 85-168 mmol/kg in the shallowest group and 250-289 mmol/kg in the deepest groups. With data from a previous study for a skate at 2,850 m included, a second-order polynomial fit suggested a plateau at the greatest depths. When data for skates (Rajidae) were analyzed separately, a sigmoidal fit was suggested. Thus, the deepest chondrichthyans may be unable to accumulate sufficient TMAO to counteract pressure; however, deeper-living specimens are needed to fully test this hypothesis.     

Temperature effects on trimethylamine oxide accumulation and the relationship between plasma concentration and tissue levels in smelt (Osmerus mordax)

Rainbow smelt (Osmerus mordax) were maintained in a long term acclimation study to elucidate temperature effects on the accumulation of trimethylamine oxide (TMAO) and to determine if the activity of trimethylamine oxidase (TMAoxi) plays a role in modulating the seasonally variable levels of TMAO. In the same experiment, the TMAO content was determined for several tissues at varying plasma TMAO concentrations. TMAO accumulation begins at 5-7 degrees C, well above that which might be normally associated with an antifreeze response. The plasma concentration reached a plateau of 20 mM as temperatures reached 0 degrees C. Plasma TMAO concentration drops to pre-accumulation levels, less than 5 mM, when fish are held at elevated temperature (8-11 degrees C) and increases when fish are chilled below ambient seawater temperatures. However, despite temperatures near or below 0 degrees C, plasma TMAO decreases after the winter season. Changes in TMAoxi activity do not correlate with TMAO levels, suggesting that the activity of this enzyme does not play a key role in regulating TMAO concentrations in smelt. For the first time in any teleost fish, tissue TMAO contents in liver, kidney, brain, and intestine were found to strongly correlate with plasma TMAO concentrations. For these tissues, the intracellular and extracellular concentration of TMAO appears to be approximately equal. Conversely, the heart and white muscle accumulate TMAO, and in the case of white muscle, intracellular concentration is maintained at a constant level of approximately 35 mmol/kg, despite fluctuating plasma concentrations over a range from 0 to over 25 mM.     

Effect of salinity on flavin-containing monooxygenase expression and activity in rainbow trout (Oncorhynchus mykiss)

In order to obtain more information about the physiological role(s) of flavin-containing monooxygenases (FMOs) in euryhaline teleost fishes, two experimental series were performed using adult and juvenile rainbow trout (Oncorhynchus mykiss). Cannulated adult trout were exposed to freshwater or 21% seawater for 48 h, whereas juvenile trout were acclimated to one of four different salinities: freshwater, 7%, 14%, or 21% during a 2-week period. FMO expression and activity were determined in red blood cells (RBC), liver, gill, kidney, gut, heart and brain. Furthermore, the content of trimethylamine oxide (TMAO; an FMO metabolite and an osmolyte) as well as urea were determined in various tissues. FMO expression and activity increased significantly and in a salinity dependent manner in osmoregulatory organs (gills, kidney and gut) in both juveniles and adult trout and, furthermore, in RBC in adults. No significant changes were observed in liver or heart. Urea content increased significantly and in a salinity dependent manner in all tissues, whereas TMAO was accumulated primarily in muscle tissue. Salinity dependent adjustment of FMO expression and activity primarily in osmoregulatory organs as well as regulation of TMAO content in muscle is consistent with previous studies showing an association of FMO with osmoregulation in euryhaline teleosts. However, the lack of a parallel increase of TMAO with urea in other tissues of fish at high salinity indicates other mechanisms of protection from intracellular urea may exist in non-muscular tissues.     

The little skate Raja erinacea exhibits an extrahepatic ornithine urea cycle in the muscle and modulates nitrogen metabolism during low-salinity challenge

Urea synthesis via the hepatic ornithine urea cycle (OUC) has been well described in elasmobranchs, but it is unknown whether OUC enzymes are also present in extrahepatic tissues. Muscle and liver urea, trimethylamine oxide (TMAO), and other organic osmolytes, as well as selected OUC enzymes (carbamoyl phosphate synthetase III, ornithine transcarbamoylase, arginase, and the accessory enzyme glutamine synthetase), were measured in adult little skates (Raja erinacea) exposed to 100% or 75% seawater for 5 d. Activities of all four OUC enzymes were detected in the muscle. There were no changes in muscle OUC activities in skates exposed to 75% seawater; however, arginase activity was significantly lower in the liver, compared to controls. Urea, TMAO, and several other osmolytes were significantly lower in the muscle of little skates exposed to 75% seawater, whereas only glycerophosphorylcholine was significantly lower in the liver. Urea excretion rates were twofold higher in skates exposed to 75% seawater. Taken together, these data suggest that a functional OUC may be present in the skeletal muscle tissues of R. erinacea. As well, enhanced urea excretion rates and the downregulation of the anchor OUC enzyme, arginase, in the liver may be critical in regulating tissue urea content under dilute-seawater stress.     

Correlation of trimethylamine oxide and habitat depth within and among species of teleost fish: an analysis of causation

Most shallow-water teleosts have moderate levels of trimethylamine N-oxide (TMAO; approximately 50 mmol/kg wet mass), a common osmolyte in many other marine animals. Recently, muscle TMAO contents were found to increase linearly with depth in six families. In one hypothesis, this may be an adaptation to counteract the deleterious effects of pressure on protein function, which TMAO does in vitro. In another hypothesis, TMAO may be accumulated as a by-product of acylglycerol (AG) production, increasing with depth because of elevated lipid metabolisms known to occur in some deep-sea animals. Here we analyze muscle TMAO contents and total body AG (mainly triacyglycerol [TAG]) levels in 15 species of teleosts from a greater variety of depths than sampled previously, including eight individual species caught at two or more depths. Including data of previous studies (total of 17 species, nine families), there is an apparent sigmoidal increase in TMAO contents between 0- and 1.4-km depths, from about 40 to 150 mmol/kg. From 1.4 to 4.8 km, the increase appears to be linear (r2=0.91), rising to 261 mmol/kg at 4.8 km. The trend also occurred within species: in most cases in which a species was caught at two or more depths, TMAO was higher in the deeper-caught specimens (e.g., for Coryphaenoides armatus, TMAO was 173, 229, and 261 mmol/kg at 1.8, 4.1, and 4.8 km, respectively). TMAO contents not only were consistent within species at a given depth but also did not vary with season or over a wide range of body masses or TAG contents. Thus, no clear link between TMAO and lipid was found. However, TMAO contents might correlate with the rate (rather than content) of TAG production, which could not be quantified. Overall, the data strongly support the hypothesis that TMAO is adaptively regulated with depth in deep-sea teleosts. Whether lipid metabolism is the source of that TMAO is a question that remains to be tested fully.     

Enzymatic urea adaptation: lactate and malate dehydrogenase in elasmobranchs

Lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) electrophoretic tissue patterns of two different orders of Elasmobranchii: Carchariniformes (Galeus melanostomus and Prionace glauca) and Squaliformes (Etmopterus spinax and Scymnorinus licha) were studied. The number of loci expressed for these enzymes was the same of other elasmobranch species. Differences in tissue distribution were noted in LDH from G. melanostomus due to the presence of an additional heterotetramer in the eye tissue. There were also differences in MDH. In fact, all the tissues of E. spinax and G. melanostomus showed two mitochondrial bands. Major differences were noted in the number of isozymes detected in the four compared elasmobranchs. The highest polymorphism was observed in E. spinax and G. melanostomus, two species that live in changeable environmental conditions. The resistance of isozymes after urea treatment was examined; the resulting patterns showed a quite good resistance of the enzymes, higher for LDH than MDH, also at urea concentration much greater than physiological one. These results indicated that the total isozyme resistance can be considered higher in urea accumulators (such as elasmobranchs) than in the non-accumulators (such as teleosts).     

Solute effects on mitochondria from an elasmobranch (Raja erinacea) and a teleost (Pseudopleuronectes americanus)

Varying osmolarity with sucrose/KCl media resulted in similar effects on the oxidation of glutamate by mitochondria isolated from the livers of an elasmobranch, Raja erinacea, and a teleost, Pseudopleuronectes americanus. In both species trimethylamine oxide (TMAO) inhibited mitochondrial oxidation of glutamate. Urea penetrated the inner mitochondrial membrane of both species and equilibrated with a ratio ureai/ureao of unity. Urea had little effect on the oxidation of glutamate in both species at concentrations as high as 760 mM. Addition of urea (urea/TMAO, 2:1) did not overcome the detrimental effects of TMAO in the mitochondria of either species. In the case of the elasmobranch, the osmolarity of the urea/TMAO media giving the optimal rate of respiration was hypoosmotic with respect to the intracellular osmolarity. The rate of glutamate oxidation steadily declined as osmolarity increased above this value. Assuming the osmotic profile obtained with the urea/TMAO (2:1) medium resembled most closely the in vivo situation, higher rates of oxidation or organic solutes at low osmolarity would help deplete the cell of these solutes and could contribute to cell volume regulation during hypoosmotic stress. It is suggested that two broad classes of intracellular solutes can be defined based on their effects on mitochondrial respiration. Solutes such as K+, C1-, and TMAO penetrate the inner mitochondrial membrane slowly or not at all. Increasing concentrations of these solutes result in lower rates of oxidation. This capacity may be important in regulating intracellular levels of organic solutes during osmotic stress. Solutes such as urea rapidly penetrate the cell and inner mitochondrial membrane reducing the mitochondrial volume changes associated with osmotic stress. The known detrimental effects of urea on protein structure may prevent its exclusive use as an intracellular osmotic effector.     

Trimethylamine oxide counteracts effects of hydrostatic pressure on proteins of deep-sea teleosts

In shallow marine teleost fishes, the osmolyte trimethylamine oxide (TMAO) is typically found at <70 mmol/kg wet weight. Recently we found deep-sea teleosts have up to 288 mmol/kg, increasing in the order shallow < bathyal < abyssal. We hypothesized that this protein stabilizer counteracts inhibition of proteins by hydrostatic pressure, and showed that, for lactate dehydrogenases (LDH), 250 mM TMAO fully offset an increase in NADH K(m) at physiological pressure, and partly reversed pressure-enhanced losses of activity at supranormal pressures. In this study, we examined other effects of pressure and TMAO on proteins of teleosts that live from 2000-5000 m (200-500 atmospheres [atm]). First, for LDH from a grenadier (Coryphaenoides leptolepis) at 500 atm for 8 hr, there was a significant 15% loss in activity (P < 0.05 relative to 1 atm control) that was reduced with 250 mM TMAO to an insignificant loss. Second, for pyruvate kinase from a morid cod (Antimora microlepis) at 200 atm, there was 73% increase in ADP K(m) without TMAO (P < 0.01 relative to K(m) at 1 atm) but only a 29% increase with 300 mM TMAO. Third, for G-actin from a grenadier (C. armatus) at 500 atm for 16 hr, there was a significant reduction of F-actin polymerization (P < 0.01 compared to polymerization at 1 atm) that was fully counteracted by 250 mM TMAO, but was unchanged in 250 mM glycine. These findings support the hypothesis. J. Exp. Zool. 289:172-176, 2001.     

Unique osmoregulatory morphology in primitive sharks: an intermediate state between holocephalan and derived shark secretory morphology

Discovery of an unusual rectal gland in the Atlantic sixgill shark Hexanchus vitulus led us to examine the rectal glands of 31 species of sharks to study diversity in rectal-gland morphology. Twenty-four of 31 species of sharks had digitiform glands (mean width–length ratio ± SD = 0.17 ± 0.04) previously assumed to be characteristic of all elasmobranchs regardless of habitat depth or phylogenetic age. Rectal glands from the family Somniosidae were kidney bean-shaped (mean width: length ± SD = 0.46 ± 0.05); whereas those from families Echinorhinidae and Hexanchidae were lobulate (mean width: length ± SD = 0.55 ± 0.06). Rectal gland width: length were different among species with digitiform morphology and lobulate morphology (ANOVA; R² = 0.9; df = 15, 386; 401, F = 219.24; P < 0.001). Histological and morphological characteristics of the digitiform morphology from deep-sea sharks were similar to those from shallow-water sharks. Histology of lobulate rectal glands from hexanchids were characterised by tubule bundles separated by smooth muscle around a central lumen. Additionally, we examined plasma chemistry of four species of sharks with digitiform rectal glands and two species with lobulate rectal-gland morphology to see if there were differences between morphologies. Plasma chemistry analysis showed that urea and trimethylamine N-oxide (TMAO) followed the piezolyte hypothesis, with TMAO being highest and urea being lowest in deep-sea sharks. Among electrolytes, Na⁺ was highest in species with lobulate rectal glands. Hexanchids and echinorhinids both have lobulate rectal glands similar to those of holocephalans, despite the more than 400 million years separating these two groups. The morphological similarities between the lobulate rectal-gland anatomy of primitive sharks and the secretory morphology of holocephalans may represent an intermediate state between Holocephali and derived shark species.     

TMAO and other organic osmolytes in the muscles of amphipods (Crustacea) from shallow and deep water of Lake Baikal

Concentrations of trimethylamine oxide (TMAO) and other 'compatible' osmolytes were analyzed in the muscle tissue of Lake Baikal amphipods (Crustacea) in relation to water depth of the freshwater Lake Baikal. Using HPLC and mass spectrometry, glycerophosphoryl choline (GPC), betaine, S-methyl-cysteine, sarcosine, and taurine were detected for the first time in freshwater amphipods. These osmolytes were frequently found in the five species studied but mixtures were too complex to be quantified. The pattern of these osmolytes did not change with respect to water depth. The TMAO concentration, however, was significantly higher in the muscle tissue of amphipods living in deep water than of those living in shallow water, which supports the hypothesis that TMAO acts as a protective osmolyte at increased hydrostatic pressure. We propose that eurybathic amphipods, exposed to raised hydrostatic pressure in the extremely deep freshwater Lake Baikal, have elevated TMAO levels to counteract the adverse effect of high pressure on protein structure. The elevated intracellular osmotic pressure is balanced by upregulating the extracellular hemolymph NaCl concentration.     

Urea tolerance of myofibrillar proteins of two elasmobranchs: Squalus acanthias and Raja tengu

Some biochemical properties of actomyosin and myosin from elasmobranchs, Squalus acanthias and Raja tengu are compared with those of a freshwater (Cyprinus carpio) and a marine teleost (Seriola quinquiradiata). Whereas Ca2+-ATPase of teleost actomyosins are more stable in the absence of urea, the reverse is true for elasmobranchs up to 1.0 M urea. In contrast to that of teleosts, the Mg2+-ATPase of S. acanthias actomyosin shows an activation in the presence of urea, where as that of R. tengu persists. Below 1.0 M urea, there is low incorporation of DTNB into thiols of elasmobranch myosins, and losses in alpha-helicity are reversible up to 5.0 M urea. The results, thus, demonstrate that for a certain concentration of urea, elasmobranch myofibrillar proteins may exhibit a group specific tolerance to urea.     

Maintenance and accumulation of trimethylamine oxide by winter skate (Leucoraja ocellata): reliance on low whole animal losses rather than synthesis

Trimethylamine oxide (TMAO) is typically accumulated as an organic osmolyte in marine elasmobranchs to levels second only to urea (which can reach >400 mM); however, little is known about the whole animal regulation of TMAO in elasmobranchs. In the present study on the winter skate (Leucoraja ocellata), we determine whether this species can maintain levels of TMAO in the absence of feeding, and if so, is this due to endogenous synthesis or low whole animal losses. Winter skates maintain plasma TMAO levels for up to 45 days without feeding. The liver displays methimazole oxidation, which is consistent with the presence of flavin-containing monooxygenase (E.C. 1.14.13.8) activity, the class of enzymes responsible for the physiological oxygenation of trimethylamine (TMA) to TMAO in mammals. However, no evidence for TMA oxygenation by winter skates was found using in vivo or in vitro techniques, indicating no significant capacity for endogenous TMAO synthesis. Fed skates displayed low, but measurable ( approximately 4-13 micromol.kg(-1).h(-1)), efflux of TMAO (plus TMA), whereas fasted skates did not. Using the loss of injected [14C]TMAO, it was determined that whole animal TMAO losses are likely <1% of whole body TMAO per day. These results demonstrate that winter skates utilize low whole animal TMAO losses, rather than endogenous synthesis, to maintain TMAO levels when not feeding.     

The Physiology and Evolution of Urea Transport in Fishes

This review summarizes what is currently known about urea transporters in fishes in the context of their physiology and evolution within the vertebrates. The existence of urea transporters has been investigated in red blood cells and hepatocytes of fish as well as in renal and branchial cells. Little is known about urea transport in red blood cells and hepatocytes, in fact, urea transporters are not believed to be present in the erythrocytes of elasmobranchs nor in teleost fish. What little physiological evidence there is for urea transport across fish hepatocytes is not supported by molecular evidence and could be explained by other transporters. In contrast, early findings on elasmobranch renal urea transporters were the impetus for research in other organisms. Urea transport in both the elasmobranch kidney and gill functions to retain urea within the animal against a massive concentration gradient with the environment. Information on branchial and renal urea transporters in teleost fish is recent in comparison but in teleosts urea transporters appear to function for excretion and not retention as in elasmobranchs. The presence of urea transporters in fish that produce a copious amount of urea, such as elasmobranchs and ureotelic teleosts, is reasonable. However, the existence of urea transporters in ammoniotelic fish is curious and could likely be due to their ability to manufacture urea early in life as a means to avoid ammonia toxicity. It is believed that the facilitated diffusion urea transporter (UT) gene family has undergone major evolutionary changes, likely in association with the role of urea transport in the evolution of terrestriality in the vertebrates.     

Freshwater elasmobranchs: a review of their physiology and biochemistry

Only 5% of elasmobranch species live in freshwater (FW) compared to more than 40% of known teleost species. The factors affecting the poor penetration of elasmobranchs into FW environments are currently unknown, however, an important consideration may be the high urea requirement of many proteins in marine elasmobranchs. Urea is an important osmolyte in marine elasmobranchs and must be reduced in dilute environments. There are three identifiable stages in the successful colonization of FW. The euryhaline marine species freely entering and leaving FW represent the initial stage of FW colonization. In this group, there is an apparent inability to eliminate all urea due to protein integrity issues and this results in energy and nitrogen losses that may constrain growth and reproduction. The second stage is represented by those species that live entirely in FW but must also retain some urea. This group also suffers from the same constraints as the first group. These two groups have kidneys and sensory organs that more closely resemble strictly marine forms. The third and final stage is represented by the Potamotrygonid stingrays where the need for urea in FW has been eliminated. Consequently nitrogen and energy losses are reduced and those sections of the kidney needed for urea conservation have been eliminated. The driving force for such modifications is a reduction in urea levels and the concomitant saving of energy needed for urea synthesis. Other physiological adaptations associated with survival in FW include giving birth to live young, the capacity of sperm to be activated in freshwater and modifications of the electrosensory system to function in a low conductivity environment. The need for many anatomical, metabolic and physiological modifications for FW existence may constrain the rapidity and hence the frequency of FW colonization, compared to the situation in the more advanced osmoregulating teleosts. Once optimally adapted to FW, recolonization of sea water by elasmobranchs is problematic due to the loss of urea synthetic capacity and renal structures for urea retention.     

Metabolic organization of the spotted ratfish, Hydrolagus colliei (Holocephali: Chimaeriformes): insight into the evolution of energy metabolism in the chondrichthyan fishes

The metabolic organization of a holocephalan, the spotted ratfish (Hydrolagus colliei), was assessed using measurements of key enzymes of several metabolic pathways in four tissues and plasma concentrations of free amino acids (FAA) and non-esterified fatty acids (NEFA) to ascertain if the Holocephali differ metabolically from the Elasmobranchii since these groups diverged ca. 400 Mya. Activities of carnitine palmitoyl transferase indicate that fatty acid oxidation occurs in liver and kidney but not in heart or white muscle. This result mirrors the well-established absence of lipid oxidation in elasmobranch muscle, and more recent studies showing that elasmobranch kidney possesses a capacity for lipid oxidation. High activities in oxidative tissues of enzymes of ketone body metabolism, including D-beta-hydroxybutyrate dehydrogenase, indicate that, like elasmobranchs, ketone bodies are of central importance in spotted ratfish. Like many carnivorous fishes, enzyme activities demonstrate that amino acids are metabolically important, although the concentration of plasma FAA was relatively low. NEFA concentrations are lower than in teleosts, but higher than in most elasmobranchs and similar to that in some "primitive" ray-finned fishes. NEFA composition is comparable to other marine temperate fishes, including high levels of n-6 and especially n-3 polyunsaturated fatty acids. The metabolic organization of the spotted ratfish is similar to that of elasmobranchs: a reduced capacity for lipid oxidation in muscle, lower plasma NEFA levels, and an emphasis on ketone bodies as oxidative fuel. This metabolic strategy was likely present in the common chondrichthyan ancestor, and may be similar to the ancestral metabolic state of fishes.     

Trimethylamine oxide, betaine and other osmolytes in deep-sea animals: depth trends and effects on enzymes under hydrostatic pressure.

Most shallow teleosts have low organic osmolyte contents, e.g. 70 mmol/kg or less of trimethylamine oxide (TMAO). Our previous work showed that TMAO contents increase with depth in muscles of several Pacific families of teleost fishes, to about 180 mmol/kg wet wt at 2.9 km depth in grenadiers. We now report that abyssal grenadiers (Coryphaenoides armatus, Macrouridae) from the Atlantic at 4.8 km depth contain 261 mmol/kg wet wt in muscle tissue. This precisely fits a linear trend extrapolated from the earlier data. We also found that anemones show a trend of increasing contents of methylamines (TMAO, betaine) and scyllo-inositol with increasing depth. Previously we found that TMAO counteracts the inhibitory effects of hydrostatic pressure on a variety of proteins. We now report that TMAO and, to a lesser extent, betaine, are generally better stabilizers than other common osmolytes (myo-inositol, taurine and glycine), in terms of counteracting the effects of pressure on NADH Km of grenadier lactate dehydrogenase and ADP Km of anemone and rabbit pyruvate kinase.     

The dogfish shark (Squalus acanthias) increases both hepatic and extrahepatic ornithine urea cycle enzyme activities for nitrogen conservation after feeding

Urea not only is utilized as a major osmolyte in marine elasmobranchs but also constitutes their main nitrogenous waste. This study investigated the effect of feeding, and thus elevated nitrogen intake, on nitrogen metabolism in the Pacific spiny dogfish Squalus acanthias. We determined the activities of ornithine urea cycle (O-UC) and related enzymes in liver and nonhepatic tissues. Carbamoyl phosphate synthetase III (the rate-limiting enzyme of the O-UC) activity in muscle is high compared with liver, and the activities in both tissues increased after feeding. The contribution of muscle to urea synthesis in the dogfish body appears to be much larger than that of liver when body mass is considered. Furthermore, enhanced activities of the O-UC and related enzymes (glutamine synthetase, ornithine transcarbamoylase, arginase) were seen after feeding in both liver and muscle and were accompanied by delayed increases in plasma urea, trimethylamine oxide, total free amino acids, alanine, and chloride concentrations, as well as in total osmolality. The O-UC and related enzymes also occurred in the intestine but showed little change after feeding. Feeding did not change the rate of urea excretion, indicating strong N retention after feeding. Ammonia excretion, which constituted only a small percentage of total N excretion, was raised in fed fish, while plasma ammonia did not change, suggesting that excess ammonia in plasma is quickly ushered into synthesis of urea or protein. In conclusion, we suggest that N conservation is a high priority in this elasmobranch and that feeding promotes ureogenesis and growth. Furthermore, exogenous nitrogen from food is converted into urea not only by the liver but also by the muscle and to a small extent by the intestine.     

Unusual organic osmolytes in deep-sea animals: adaptations to hydrostatic pressure and other perturbants

Shallow-living marine invertebrates use free amino acids as cellular osmolytes, while most teleosts use almost no organic osmolytes. Recently we found unusual osmolyte compositions in deep-sea animals. Trimethylamine N-oxide (TMAO) increases with depth in muscles of some teleosts, skates, and crustaceans (up to 300 mmol/kg at 2900 m). Other deep-sea animals had high levels of (1). scyllo-inositol in echinoderms, gastropods, and polychaetes, (2). that polyol plus beta-alanine and betaine in octopods, (3). hypotaurine, N-methyltaurine, and unidentified methylamines in vestimentiferans from hydrothermal vents and cold seeps, and (4). a depth-correlated serine-phosphate osmolyte in vesicomyid clams from trench seeps. We hypothesize that some of these solutes counteract effects of hydrostatic pressure. With lactate dehydrogenase, actin, and pyruvate kinase, 250 mM TMAO (but not glycine) protected both ligand binding and protein stability against pressure. To test TMAO in living cells, we grew yeast under pressure. After 1 h at 71 MPa, 3.5 h at 71 MPa, and 17 h at 30 MPa, 150 mM TMAO generally doubled the number of cells that formed colonies. Sulfur-based osmolytes which are not correlated with depth, such as hypotaurine and thiotaurine, are probably involved in sulfide metabolism and detoxification. Thus deep-sea osmolytes may have at least two other roles beyond acting as simple compatible osmotica.     

Carbon and nitrogen discrimination factors for elasmobranch soft tissues based on a long-term controlled feeding study

The foraging ecology of elasmobranchs (sharks, skates, and rays) is difficult to study because species have spatially and temporally diverse diets. Many diet and habitat preference studies for mammals, birds, and teleosts use stable isotope analysis, but interpretations are limited for elasmobranch studies because taxon-specific isotope discrimination factors from a controlled experiment are unavailable. Trophic discrimination factors for plasma, red blood cells, and muscle were determined from an experiment with leopard sharks (Triakis semifasciata) fed a constant diet of squid over 1000?days. The ??¹³C values for shark tissues at equilibrium with the squid diet did not vary significantly among individuals, but plasma and red blood cell ??¹⁵N values differed significantly among individuals and sampling day. Individual variation of muscle ??¹⁵N averages was observed and likely related to growth. Overall, carbon and nitrogen discrimination factors corresponded to previous studies featuring high-protein diets and carnivorous taxa. The muscle-to-diet discrimination factors from the controlled feeding study were applied to blue sharks (Prionace glauca) and smooth hammerhead sharks (Sphyrna zygaena) caught offshore from Baja California, Mexico. This case study demonstrates the potential of stable isotope analysis to illuminate differences in foraging patterns between elasmobranch species.